Project Summary Rift Valley fever virus (RVFV) (Phenuiviridae: Phlebovirus) is an emerging mosquito-transmitted virus with a high probability of introduction to new areas, including the United States. Significant questions remain regarding the inter-epidemic maintenance of RVFV in endemic countries, and the ecological importance of North American mosquitoes in the establishment and transmission of RVFV. In particular, RVFV is currently believed to be maintained in natural mosquito vector populations through vertical transmission, however published laboratory evidence documenting this phenomenon is completely lacking. We recently demonstrated high RVFV dissemination rates and infection of ovarian tissues in both Ae. aegypti and Cx. tarsalis mosquitoes orally exposed to an epidemic strain of RVFV from Kenya. The infection rate of the 1st instar larvae of Cx. tarsalis mosquitoes was 87% (Bergren, preliminary data). This is the first time transovarial transmission of RVFV has been experimentally demonstrated in any mosquito species. Interestingly, transstadial transmission of RVFV from eggs to F1 adult life stages was inhibited in Ae. aegypti due to an unknown mechanism. This project will address the hypotheses that the efficiency of transstadial transmission of RVFV is mediated by larval habitat temperature (Aim 1), as well as mosquito anti-viral effectors and Wnt signalling during immature developmental stages (Aim 2). For Aim 1, both Cx. tarsalis and Ae. aegypti mosquitoes derived from RVFV- infected parental mosquitoes will be reared at low (18C) standard (28C), and high (35C) temperatures. Transstadial virus persistence in each mosquito species will be quantified by qRT-PCR and plaque titration of 1st instar, 4th instar, pupal, and F1 adult mosquitoes reared at each temperature. Statistical analysis of infection rates as well as virus titer in each life stage as a function of mosquito species and temperature will inform whether or not larval rearing temperature significantly influences transstadial persistence of RVFV. To support these environmental data, RNA-Seq will be employed in Aim 2 to identify the underlying molecular pathways contributing to our preliminary phenotypic observations. Differential gene expression will be analyzed in individual mosquitoes of each species throughout the developmental process. The function of top-candidate genes during RVFV transstadial transmission will be confirmed by specific double-stranded RNA knock-down in mosquito larvae. The data generated in these studies will collectively elucidate environmental and molecular drivers of RVFV persistence in North American mosquito populations. This project will also directly address shortcomings in current predictive models regarding the environmental persistence and establishment potential of RVFV in mosquitoes.